Fowlpox virus non-essential regions

ABSTRACT

The invention relates to DNA from fowlpox virus (FPV) providing a non-essential region for the insertion of foreign genes thereinto and thence the construction of a vector for homologous recombination with a wild type FPV, whereby the resulting recombinant FPV can be used for vaccination of animals, especially chickens. In this invention, the non-essential region consists substantially of a length of DNA from the long unique sequence of the terminal inverted repeat (TIR) of FPV or from the region at FPV which corresponds approximately to the HindIII D fragment genes D8 and D9 in vaccinia virus.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention is in the field of recombinant DNA technology and relatesto fowlpox virus as a vector for foreign DNA.

2. Description of the Prior Art

Several viruses with DNA genomes have been used to carry and expressgenes from other viruses or other species. Viruses used in such a wayare known as `vectors` and genes, other than their own, expressed insuch a way are referred to as `foreign genes`. One of the primaryrequirements for a virus to be used as a vector in this manner is asuitable site for insertion of the foreign gene. If insertion of a geneinto a site in the virus causes disruption of some function essentialfor growth, then such a site could not be used. Suitable sites are thoseat which an insertion does not disrupt any functions or those whosefunctions are not essential for viral growth and therefore can bedisrupted with impunity. Such sites are known as `non-essentialregions`. The phrase `non-essential` in this context means non-essentialfor growth under at least some conditions in which the virus can begrown in vitro and under at least some conditions in which it survivesin vivo.

Examples of viruses which have been used as vectors by virtue of thefact that they contain non-essential regions are orthopoxviruses,adenoviruses and herpesviruses, although the regions used may bedifferent in each case. Vaccinia virus (VV), which has been used as avector, is an orthopoxvirus, a member of the pox virus family.Fowlpoxvirus (FPV), the subject of this patent application, is also apox virus, but is a member of a different genus, the avipoxviruses. VVcan be grown in tissue culture, and foreign genes can be inserted intothe viral genome during this process. Several regions of VV have beenfound to be non-essential in vitro in more than one tissue-culturesystem. These include: large regions towards the left hand end, B. Mosset al., J. Virology 40, 387-395, (1981) who describe a mutant VV havinga deletion 6.4 megaDaltons from the left-hand end; D. Panicali et al.,J. Virology 37 1000-1010, 1981) who describe a mutant VV having adeletion starting 6.85 megaDaltons from the left-hand end; the thymidinekinase (TK) gene, D. Panicali and E. Paoletti, Proc. Natl. Acad. Sci.USA 79, 4927-4931 (1982); M. Mackett et al., J. Gen. Virol. 45, 683-701,(1982); and the vaccinia growth factor (VGF) gene, R. M. L. Buller ital., J. Virology 62, 866-974 (1988). Sites such as these might also benon-essential for growth in vivo. However in the case of both the TK andVGF gene, it has been found that although growth in tissue culture isunaffected or only slightly affected by insertion into the gene, growthand virulence in vivo are markedly affected, R. M. L. Buller el al.,Nature 317, 813-815 (1985) and loc. cit. (1988), showing that thesegenes are not completely non-essential for growth of the virus in vivo.This in vivo attenuation may however be useful if it reduces unwantedpathogenic effects of the virus and accordingly growth in vivo withaccompanying attenuation is a valid growth condition for the purpose ofthis invention.

Some sites are essential in some tissue culture systems andnon-essential in others. For example there is a gene in VV which isessential for replication in human cells but which is non-essential inchicken embryo fibroblast cells, S. Gillard et al., Proc. Natl. Acad.Sci. USA 83, 5573-5577 (1986). Another gene in the related orthopoxviruscowpox virus is essential for growth in chinese hamster ovary cells butnot for growth in chicken embryo fibroblast cells, D. Spehner et al., J.Virology 62, 1297-1304 (1988). These examples show that differences inthe tissue culture systems can affect which regions are non-essential.

The VV genome is far from being completely mapped and relatively littlehas been published about the FPV genome. It is known that FPV has a TKgene which has about 60% homology with the VV TK gene at the amino acidlevel , D. B. Boyle et al., Virology 156, 355-365 (1987). Like the VV TKgene, it serves as a non-essential region for homologous recombinationunder at least some conditions: see PCT Application WO 88/02022 (CSIRO).Using the E. coli xanthine-guanine phosphoribosyl transferase (Ecogpt)gene as a dominant selectable marker, in conjunction with the VV "7.5"promoter, the influenza haemagglutinin (HA) gene was inserted into a TKregion of FPV and the FPV grown in chicken embryo skin cells. Expressionof the HA gene was demonstrated by the binding of HA antibodies andlabelled protein A to plaques of recombinant FPV.

The genome of FPV is known to have other similarities to VV in someregions. For example the DNA polymerase genes are closely related,having 42% homology at the amino acid level, M. M. Binns et al., NucleicAcids Research 15, 6563-6573 (1987). On the other hand, F. M. Tomley, ina talk at the 6th Workshop on Poxvirus/Iridovirus, Cold Spring Harbor,N.Y., Sep. 24-28, 1986, described an attempted correlation of an 11.2 kbfragment of FPV DNA, located near one end of the genome, with VV DNA.While she reported 25% amino acid homology between a gene predicting a48 kd polypeptide in FPV and a gene in VV coding for a 42 kdpolypeptide, no other match of any significance was found. More detailof this work is given in F. M. Tomley et al., J. Gen. Virol. 69,1025-1040 (1988).

F. M. Tomley et al, quoting M. Mackett and L. C. Archard, J. GeneralVirology 45, 683-701 (1979), also state at page 1038 that the terminalportions of orthopoxvirus genomes are less well conserved than thecentral region and also say that the terminal regions, of around 30 to35 kb in length, are thought to encode genes which determine specificvirus characteristics such as host range, virulence, tissue tropism andcytopathogenicity and are thus less likely to be conserved.

The terminal region of VV is known to be of possible interest inrelation to non-essential regions. Thus, M. E. Perkus et al., Virology152, 285-297 (1986), have found that deletions in the VV genome occur inthe presence of bromoxydeuridine, one of which extended from aboutnucleotides 2700 to 24100 from the left-hand end and that the deletionmutant virus is viable in tissue culture (cell type not clearly stated).Some information has been released about the very terminal region ofFPV, in a poster by J. I. A. Campbell displayed at the InternationalPoxvirus Workshop, Cold Spring Harbor, N.Y., Sep. 9-13, 1987. (Theposter gives more information than does the abstract published in"Modern Approaches to New Vaccines including Prevention of AIDS", CSH1987, page 45). Like VV and cowpox virus, FPV has terminal sequenceswhich are inverted repeats of each other and are covalently cross-linkedtogether. A part of the terminal inverted repeat (TIR) sequence wasdigested with BamHI and cloned. The BamHI fragment was described ashaving a length of 6.3 kb. The poster describes the general layout ofthe sequences, which from left to right comprise a short unique region,a set of tandemly repeated sequences and then a long unique regioncontaining three possible open reading frames. The summary notes thatthis FPV terminal fragment shares the general pattern of nucleotidesequence with the terminal fragments of VV and cowpoxvirus, but alsonotes some marked differences. The sequence of part of the VV TIR,namely from approximately nucleotides 6800 to 9000 from the left-handend, VV i s reported by S. Venkatesan et al., J. Virology 44, 637-646(1982).

Large differences between the DNA sequence of VV and FPV are to beexpected, since the FPV genome is estimated to be at least one thirdlonger than that of VV. It seems likely, from the present knowledge ascited above, that many of the differences between the genomes of FPV andVV will be nearer to the termini than to the center of the genome. Thus,information about the terminal region of VV is of limited interest inrelation to FPV.

It has been a problem to locate a well-defined non-essential regionother than the TK gene, in fowlpox virus. Very recently (Apr. 20, 1989),in PCT application publication No. WO89/03429 (Health Research Inc.),FPV recombinants have been described, but the non-essential regionsmentioned therein are not all well defined. The recombinants weregenerated merely by cleaving the FPV gene into fragments and trying thefragments with test constructs to see whether the foreign gene undertest was expressed.

The VV HindIII-D fragment is one of the best characterised regions ofthe virus. The sequence of this 16060bp fragment has been determined,Niles et al., Virology 153, 96-112 (1986), and thirteen genes (D1-D13)have been identified.

Several temperature-sensitive mutants have been mapped to genes withinHindIII-D and fine mapping has assigned these to D2, D3, D5, D6, D7, D11and D13, Seto it al., Virology 160 110-119 (1987). These genes aretherefore essential for virus replication. It is not known whether theremaining genes within the VV HindIII-D fragment are essential ornon-essential for virus growth. Recent experiments have suggested(though not proved) that D8, which encodes a transmembrane protein,might be non-essential for propagation of vaccinia virus in tissueculture, Niles and Seto, Journal of Virology 62 3772-3778 (1988). Inthese experiments, a frameshift mutation was introduced into thecarboxy-end of D8 which removed the carboxy-terminal 56 amino acids ofD8. Virus containing this mutation had growth rates indistinguishablefrom those of wild type virus.

It was not predictable, however, whether the D8 gene would occur infowlpox virus and if so whether it would be non-essential. Moreover,there was the problem of how to locate the D8 gene in the FPV genomewhich is relatively unmapped and is much larger than that of VV.Consequently, examination of the vaccinia Virus HindIII-D fragment didnot indicate how to find further non-essential regions within FPV.

SUMMARY OF THE INVENTION

It has now been found that the three open-reading frames (ORFs) whollywithin the above-mentioned BamHI fragment of the terminal invertedrepeat (TIR) are non-essential regions which can serve as sites forhomologous recombination events whereby a foreign gene can be introducedinto FPV (refer to FIG. 1). These ORFs are characterised in that theyhave lengths of approximately 670, 370 and 300 nucleotides and fallwithin the long unique sequence (LUS) of the TIR within the BamHIfragment. The BamHI fragment has been found to have a length of about6.3 kb and the long unique sequence begins at approximately nucleotide4100, numbering from the left-hand end. (As to nucleotide numbering,please refer to the cautionary note in the discussion of FIGS.15(a)-15(c) below. The lengths of the ORFs can also be expressed interms of the approximate molecular weights of the polypeptides whichthey are predicted to encode, namely 26.0, 13.9 and 11.5 kilodaltons.This nomenclature will be used hereinafter for identification purposes.The 26.0 and 11.5 genes are coded for on the same strand and in thatorder when reading the fragment left to right from the 5' end towardsthe BamHI site, the 11.5 gene having its 3' near the BamHI site (seeFIG. 3). The 13.9 gene is encoded on the complementary strand, readingright to left, but within a region distinct from and lying in betweenthat coding for the 26.0. and 11.5 genes on the first strand.

Extrapolation from this and other findings leads to the reasonablesuppositions that the whole of the long unique sequence of the TIR is anon-essential region, including sequence lying to the immediateright-hand end of the BamHI site, i.e. within the 11.2 kb fragmentsequenced by F. M. Tomley et al., loc. cit. (1988). The TIR has a lengthof about 10 kb, i.e. 10-6.3 =3.7 kb lies within the 11.2 kb fragment.Thus, the long unique sequence extends to the right from aboutnucleotide 4100 over the remaining lengths of TIR, of about 6 kb.

Accordingly, in an important aspect the present invention provides arecombination vector for use in producing a recombination fowlpox virus(FPV) by homologous DNA recombination, said recombination vectorcomprising a cloning vector containing a non-essential region (NER)sequence comprising at least part of the long unique sequence (LUS) ofthe terminal inverted repeat (TIR) of FPV, said NER being interrupted bya foreign gene or genes and by a sequence or sequences for regulatingthe expression of each foreign gene present. Preferably the NER iswithin part of the long unique sequence (LUS) lying within the 6.3 kbBamHI fragment. In a more particular aspect, the NER consists of atleast a homologously recombinable length of an open reading frame (ORF)having a length of approximately 670, 370 or 300 nucleotides and fallingwholly within that part of the LUS within the BamHI fragment of the TIR.However, we have also found that some isolates of the same strain of FPVdo not have a lengthy sequence of over 200 bp of DNA spanning the BamHIsite, which is an important pointer to the non-essentiality of thesequence spanning that site. It is therefore inappropriate to use theterminology "BamHI fragment" in a formal definition applicable to FPVgenerally. Accordingly, these ORFs are better defined as lying withinthe first 2.2 kb of the LUS after the tandem repeats, that is to saywithin 2.2 kb of its external end. NERs within this 2.2 kb arepreferred. The above preferences for location of NERs apply also to theflanking sequences contained in the recombination vector and thisinvention, i.e. all the homologously recombinable sequence which flanksthe foreign gene(s) and their regulatory sequence(s).

We have also found certain other non-essential regions of fowlpox virus.Whereas those described above are within part of the terminal invertedrepeat, these further regions are in a more central part of the FPV DNAmolecule, namely within or adjacent to one end of a 6.3 kb fragment ofan EcoRIl ibrary of FPV, the genes of which show significant degrees ofhomology with certain genes of the vaccinia virus (VV) HindIII-Dfragment.

This second aspect of the present invention has its origins in a randomcloning of parts of the FPV genome, sequencing of the random clones anda search in all six reading frames for possible open reading frameswhich might predict genes. The sequences thus generated were comparedwith vaccinia virus (VV) protein sequences. It so happened that degreesof homology were noted to some of the VV proteins D1-D13, includingapproximate matches to VV D1, D5, D9, D10 and D13. It was thereforedecided provisionally to adopt the "D" terminology for the FPV geneswhich showed some degree of homology with the VV genes. An M13 clone ofFPV matching VV D9 was used as a probe to identify clones within anEcoRI library of FPV. One such clone, designated pMB379, containing a6.3 kb insert, was sequenced. This clone contained part of the D9 geneand all of D10, D11, D12 and D13. (The last four genes showed 40%, 62%,53% and 55% identity of amino acids encoded to their VV homologues). Inorder to clone D7 and D8 genes, a DraI library of FPV DNA cloned in theplasmid pUC13 Sm 1 was constructed. Colonies were probed with aprime-cut probe derived from an M13 clone designated M379-27S (whichlies within the FPV D9 gene) and one positive colony, containing aplasmid designated pMB389, was studied further. pMB389 was found tocontain the "carboxy"-end of the D6 gene, entire D7, D9, D10 genes andthe "carboxy"-end of the D11 gene. (D11 runs in the reverse direction toD10, so the carboxy end is that nearer to D10. The "carboxy" end is thatwhich is the 3'- end of the coding strand, corresponding to the carboxyend of the predicted polypeptide). There was no identifiable homologueof the VV D8 gene, although another gene was present between D7 and D9.This has been termed "D8" as a matter of convenience. In a particularlyunexpected finding, the copy of D9 present in both pMB379 and pMB389possessed frameshift mutations when compared with its VV counterpart,thus making it unlikely that D9 is expressed in FPV.

It has now been found that the D8 and D9 genes of FPV are non-essentialgenes which can be used for the insertion of foreign DNA. Since theintergenic regions can also be considered non-essential, it follows thatin principle the whole of the region following the carboxy-end of D7 andup to the carboxy end of the D9 gene is a non-essential region (NER).(The genes D7, D8, D9 and D10 all run in the same direction). However,as will be explained hereinafter, the NER does not run right up to thecarboxy-end of the D9 gene, but stops short thereof in order to avoidpossible interference with the D10 gene, which overlaps with the D9, andthe promoter of the D10 gene. This NER will be referred to hereinafterfor brevity as the D8-D9 NER.

Accordingly, the present invention further provides a recombinationvector as set forth above but wherein the NER is such a carboxy end-D7to near carboxy end-D9 region NER (but excepting the D10 gene and D10promoter region).

In a second important aspect the invention provides a recombinantvector, e.g. a plasmid, containing a NER sequence (TIR or D8-D9) asdefined above. Such a vector is useful for preparing the above-definedrecombination vector.

Thirdly, the invention includes a DNA molecule which consistssubstantially or essentially of a NER sequence as defined above with orwithout some homologously recombinable flanking sequence, say(arbitrarily) up to 1000 bp per end at either or both ends thereof inorder to distinguish such a molecule from the genomic DNA of FPV.

Fourthly, the invention covers a host, appropriate to the vector,harbouring any of the vectors of the invention, i.e. the recombinationvector or any intermediate vector which contains the novel NER sequence.

Fifthly, the invention embraces a recombinant FPV produced by homolgousrecombination of a parent FPV with the insert DNA of a recombinationvector defined above.

Sixthly, the invention includes animal, especially chicken, cellsinfected with the above-identified recombinant FPV, a process of invitro culture of said cells and, where patent law permits, a method ofvaccinating a responsive animal, especially a chicken, with therecombinant FPV.

In a more particular aspect the recombination vector of the inventioncomprises in order:

(1) a first homologously recombinable flanking sequence,

(2) a sequence within a first portion of the non-essential region,

(3) promoter DNA,

(4) a foreign gene transcribably downstream of the promoter (wherebywhen the fowlpox virus RNA polymerase binds to the promoter it willtranscribe the foreign gene into mRNA),

(5) a sequence within a second portion of the same non-essential region,the first and second sequences being in the same relative orientation asare the first and second portions of the non-essential region within theviral genome, and

(6) a second homologously recombinable flanking sequence.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic illustration of a homologous recombination processof the invention;

FIG. 2 is a map of the BamHI terminal fragment of FPV DNA showing thelocation therein of the beginning of the long unique sequence of theTIR;

FIG. 3 is a DNA map of the FPV TIR showing three non-essential regionsequences useful in the present invention and other ORFs, together witha map of the VV TIR showing the different ORFs found in VV;

FIG. 4 to 7 illustrate schematically various general methods ofconstruction of recombination plasmids in accordance with the invention;and

FIG. 8 illustrates schematically the construction of specificrecombination plasmids of the invention containing non-essential regionsequence of FPV into which a foreign gene is inserted.

FIG. 9 is a map of the genes D6 to D13 and an adjacent gene A2 showingthe genes cloned in plasmids pMB379 and 389 and important restrictionsites in these plasmids;

FIG. 10 illustrates schematically the construction of specificrecombination plasmids of the invention containing sequence within theD8, D9 and D10 genes (D8, D9 according to the invention; D10comparative) into which a foreign gene is inserted.

FIG. 11 depicts construction of plasmid pBGF1;

FIG. 12 depicts construction of plasmid pEFF2;

FIG. 13 depicts construction of plasmid pBHN1;

FIG. 14 depicts construction of plasmid pEFH3; and FIGS. 15(a) to 15(c)show the DNA sequence of the BamHI fragment starting from the left handend, reading in the 5' to 3' direction.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

Referring first to the terminal inverted repeat (TIR) NERs and to FIG.2, the BamHI fragment of the TIR of FPV has a length of about 6.3 kb. Itcontains a short unique sequence of length 0.23 kb, followed by a regionof approximate length 3.87 kb which contains tandemly repeated sequencesand then a long unique sequence of length 2.18 kb.

The approximate location of the genes providing the TIR NER DNA for usein the invention within the BamHI fragment is shown in the map of FIG.3. The arrows show that the 13.9 gene is on a complementary strand. Fromthis map and from sequence information it will be seen that these geneshave no known near counterpart in VV. The BamHI site is believed to bepart of a further non-essential region for the reasons explained above.A total of five ORFs have been found in the long unique sequence of theTIR. Those which appear to encode polypeptides by virtue of theirdistinctive FPV codon bias occur as follows:

    ______________________________________                                        Location                                                                      (Nucleotides        No.                                                       numbered from                                                                           No.       amino acids                                                                             M.w. of Unique                                  left-hand end)                                                                          nucleotides                                                                             encoded   polypeptide                                                                           site                                    ______________________________________                                        4125-4790 666       222       26.0 kd Bgl II                                  5261-5629C                                                                              369       123       13.9 kd Nco I                                   5883-6185 303       101       11.5 kd Spe I                                   ______________________________________                                    

C=occurs on the strand complementary to that shown in the nucleotidesequence below.

Besides the three ORFs designated 26.0, 13.9 and 11.5, there are twoshorter ORFs on the complementary strand. Since, however, neither ofthese ORFs exhibit the required FPV codon bias it is unlikely that theycode for genes.

FIGS 15(a)-15(c) show the DNA sequence of the BamHI fragment, startingfrom the left-hand end and reading in the usual 5' to 3' direction,together with predicted polypeptides encoded. It should be borne inmind, however, that the sequencing of the tandemly repeated region haspresented difficulties and that the number of nucleotides within thisregion might be subject to some revision. This is not a matter of anysignificance to the present invention, which is not concerned with thetandemly repeated region. It follows that the long unique sequence doesnot necessarily start at about nucleotide 4110 in absolute numbering andthe nucleotide numbers are therefore to be treated as merely guidance tothe reader to enable him to identify the ORFs referred to and to beconstrued accordingly in the description and claims. It will beappreciated that the sequence shown in FIGS. 15(a) to 15(c) definitiveof FPV generically, since there will inevitably be small variationsbetween different types or strains of FPV, some of which might take theform of deletions or insertions, and any variant NER compatible with thedesired homologous recombination is usable herein. In particular, DNAhaving a NER sequence based on ORFs having a sequence showing at least,say, 90% homology with the sequence set forth above is a preferredfeature of the invention.

Beyond the BamHI site (GGATCC) the nucleotide sequence continues as perF. M. Tomley et al. loc. cit. (1988) at page 1029, nucleotide 6275 shownabove being the first of the BamHI recognition sequence and No. 1 inTomley et al.'s numbering. While there are no ORFs spanning the BamHIsite, the sequence extending at least as far as the first 160 bp intothe (11.2 kb fragment of Tomley et al. is demonstrable non-essential,while further sequence thereafter is likely to be.

The NERs of the invention can be used to insert foreign genes into FPV.It is desirable that the gene be stably incorporated so that theresultant recombinant FPV can be repeatedly cultured in tissue cultureon a large scale. Evidence to date is that the 13.9 gene is less stablethan the other two.

The foreign gene can be inserted at any location within the NER sequenceof the recombination plasmid. Preferably it is not so excessively nearone end or the other as not to provide a site long enough for homologousrecombination with the corresponding NER of the wild type FPV. However,it is possible to allow homologous recombination to occur outside theNER by providing, in the recombination vector, flanking sequence lyingbeyond either or both ends of the NER. It is probably not usuallybeneficial to use flanking sequences which are heavily tandemlyrepeated, since this will increase the chances of unwanted recombinationevents. Thus, it is suggested that flanking sequences runningextensively beyond the external end of the LUS into the tandem repeatregion are best avoided.

Referring now to the D8-D9 NER and to FIG. 9 of the drawings, theplasmids pMB379 and pMB389 have FPV DNA inserts which overlap in the D9and D10 gene region. By sequencing these inserts a long nucleotidesequence has been obtained which runs from the 3'-end of D6 through toD13 and beyond that to the adjacent A2 gene.

In the sequence shown below, those parts which are not relevant to thepresent invention have been truncated. The restriction sites Dral(TTTAAA), EcoRI (GAATTC), BclI (TGATCA), AsuII (TTCGAA) and BulII(AGATCT) used in the cloning and construction of plasmids for testingout non-essential regions are underlined. The amino acid sequences ofpredicted genes are given in the single letter code. The D9 gene, beingconsidered to be unexpressed in FPV, is not given an amino acidsequence, but is considered to run from nucleotides 1298 to 1984. Startand stop codons have been underlined. The sequence shown encompasses thecarboxy end of the D7 gene and the carboxy end of the D11 gene. (Sincethe D11 gene has an open-reading frame running in the reverse directionto the others, its near-D10 end is that encoding the carboxy terminus ofthe putative D11 protein). The sequence shows the DraI site in the D11gene (see also FIG. 9).

It will be seen that the D10 gene starts within the end of the D9 gene,being within a different reading frame. The D10 promoter is thereforealso within the D9 gene. The non-essential region is therefore definedas extending up to the beginning of the D10 promoter region, which isestimated at approximately 35 bases before the ATG start codon for D10,i.e. at around nucleotide 1936. ##STR1##

There are some preliminary indications that the insertion of a foreigngene in the D10 gene is unstable. Accordingly the D10 gene is not a NERfor the purposes of the invention.

Referring now to all the NERs of this invention, the recombinationvector can contain a reasonable length of homologously recombinableflanking sequence to each side of the NER, preferably at least 100nucleotides, more preferably at least 250 nucleotides and still morepreferably at least 500 nucleotides. On the other hand, in the interestsof not making the insert for the recombination vector too large, it isoften desirable to limit such flanking sequence outside the NER to, say,not more than 1000 bp.

Incidentally, precise homology of the flanking sequence and the NER isnot necessarily required. Remarks to that effect in WO 89/03429 applyhere also.

In preparing recombinant FPV of the invention, the first requirement isfor a promoter. The well-known vaccinia virus P7.5 promoter the H6promoter (see the cited PCT application publication No. WO 89/03429) orthe P11 promoter, can be used, as these are accepted by the FPV ascompatible. Preferably, however, a FPV promoter is used. Certainpromoters have been described in the prior PCT patent applicationpublication No. WO 89/03879 (National Research Development Corporation),the contents of which are herein incorporated by reference. The "4b" and"13.2K" promoters therein are especially preferred.

The invention has particular relevance to poultry, especially chickens.For this purpose, any foreign gene relevant to improving the conditionof poultry could be inserted into the fowlpox virus. Preferably the genewill be one appropriate to an in vivo sub-unit vaccine, for example oneor more genes selected from Infectious Bronchitis Virus (IBV),Infectious Bursal Disease virus, Newcastle Disease Virus (NDV), Marek'sdisease virus, infectious laryngotracheitis virus and genes encodingantigenic proteins of Eimeria species. Particular genes of interest arethe spike genes of IBV and the HN and F genes of NDV as described in PCTpatent application publication No. WO 86/05806 and European PatentApplication Publication No. 227414A (both National Research DevelopmentCorporation). In order for the foreign gene to be correctly translatedin vivo it is necessary for the foreign gene to have its own ATG startcodon inserted in the region just following the promoter.

More than one foreign gene can be expressed in a single poxvirus. It ispossible to arrange for mRNA for two foreign genes to be transcribed indifferent directions when inserted in a single non-essential region.Such a "back to back" construction is shown in Example 7. Also,individual foreign genes can be inserted in individual NERs describedherein. While fusion protein genes can, of course, be made, it isordinarily preferable that each gene be under control of a separatepromoter.

The promoter and foreign gene then have to be inserted into thenon-essential region (NER) of the FPV genome. The procedure ofhomologous recombination, illustrated by FIG. 1 of the drawings,provides a way of doing so. A fragment of genomic DNA containing the NERis sub-cloned in a cloning vector. A construct is then made, in thecloning vector, comprising part of the NER followed by the promoterfollowed by the foreign gene, followed by a further part of the NER inthe same orientation as the first part. This construct, in anappropriate vector, forms the recombination vector which is used totransfect the FPV, e.g. by the calcium phosphate method, wherebyrecombination occurs between the NER sequences in the recombinationvector and the NER sequences in the FPV. The FPV then automaticallyre-packages this altered genome and the thus altered FPV (recombinantFPV) is part of this invention.

The above-described recombination vector can be prepared in manydifferent ways, the requisite elements being introduced in any order.Referring to FIG. 4, a non-essential region defined above for use in theinvention possessing (say) two restriction sites A, B is inserted in anappropriate vector, which by way of illustration only will be describedas a plasmid. In another plasmid having the same (or ligatablycompatible) restriction sites A, B, a construct is made of promotersequence followed by the foreign gene sequence. It is of courseessential that this construct is made so that the mRNA transcriptionwill begin at or before the start codon of the foreign gene. Since it istime-consuming to determine precisely where the mRNA transcription startis effected by any particular promoter, it is convenient simply toinsert, say, 100 or more preferably 150 base pairs of promoter DNAimmediately preceding the FPV gene which it normally promotes, to ensuregood working of the promoter.

The restriction sites A, B are located in the plasmid DNA flanking theFPV promoter DNA and the foreign gene. Of course, A can be within thepromoter DNA if it falls within a non-functional portion thereof. WhileA and B can be two different restriction sites, they can, of course, beone and the same. They can be sticky- or blunt-ended sites and can beprepared artificially, e.g. by filling in with additional nucleotides orchewing back, in ways well known in the recombinant DNA field.Conveniently A and B are or are converted into a single blunt-ended site(C) and then allowed to ligate into a single blunt-ended site (C) withinthe NER. Care will have to be taken, of course, to select sites whichare unique in the plasmid DNA to prevent ligation of other sequences ofDNA from occurring. In the Example herein, unique sites for SpeI, NcoIand BglII in the NERs have been used and have been filled in to providea single blunt-ended site for insertion of the promoter-gene construct.

DNA from the two plasmids are ligated together in vitro and thentransformed into the host, to produce the final recombination plasmid ofthe invention.

FIG. 5 illustrates another method of preparing recombinant vectors. Inthis method one first prepares a construct comprising a first part ofthe NER, followed by the promoter, followed by a short sequence ofnucleotides containing at least one cloning site for introduction of aforeign gene, followed by a second part of the NER which will almostinevitably be in the same orientation as the first part. Of course,virtually any length of DNA would provide a cloning site suitable insome way or other for introducing a foreign gene. Preferably theseconstructs contain a multiple cloning site, that is to say a length ofDNA containing the sites of a variety of different restriction enzymes,for example at least ten. Such a construct then has versatility, sinceit will then be much easier to restrict DNA flanking almost any foreigngene at sites close to each end thereof and insert the foreign gene intothe multiple cloning site illustrated in FIG. 5. Only two sites X, Yhave been shown, for simplicity and these can be filled in or chewedback, as desired, to give identical blunt-ended sites (Z, replacing X,Y). In the final recombination plasmids the promoter DNA will beseparated from the foreign gene by a portion of the multiple cloningsite, but this will not adversely affect the transcription of the MRNAin the final virus.

In either method of construction the NER is split by the promoter andforeign gene. It is, of course, not essential that it be split in acentral region. Nor is it essential that the second part thereofconstitute the entire balance or remainder of the NER. As explainedabove, so long as there exists within or at each end of the NER containsa long enough stretch of DNA for homologous recombination, it does notmatter that a part of the NER might be excised somewhere in between orthat additional (irrelevant) DNA be inserted in preparing therecombination plasmid, or even that the NER is only a few bp in length(but it should not normally be less than about 20 bp long or there wouldbe practical difficulties in inserting the foreign DNA).

FIG. 6 illustrates another method of preparing a recombination plasmid.The NER plasmid is restricted as before (a blunt-ended site or stickyended site filled in to become blunt-ended, C, is illustratedarbitrarily for this Figure). The other plasmid contains a marker genewith its own promoter and a foreign gene with its promoter in opposedorientations. Different promoters should be used to avoid thepossibility of an incorrect recombination. When these plasmids arelegated together the marker is linked to the foreign gene. The presenceof the marker in the recombinant FPV is therefore likely to indicate asuccessful incorporation of the foreign gene into FPV. Conveniently themarker is readily detectable by a colour, fluorescent orchemiluminescent reaction. The beta-galactosidase or lacZ gene can beinserted into the NER and recombinants detected by the blue plaquesgenerated when the 5-bromo-4-chloro-3-indolyl-D- galactopyranoside(X-gal) substrate is present in the growth medium. This technique, usingthe lacz gene, has been described in relation to VV by S. Chakrabarti etal., Molecular and Cellular Biology 5, 3403-3409 (1985).

Preferably the marker gene is also selectable (or it is replaced by aselectable gene or two separate genes, one marker and one selectable areincluded). An example of a selectable gene is the Ecogpt gene describedin PCT Application WO 88/02022, already cited, and also by F. G. Falknerand B. Moss, J. Virology 62, 1849-1854 (1988).

It is also useful when preparing a recombination plasmid to know thatthe foreign gene is likely to have been successfully inserted into theNER. One way of checking is to adopt the procedure shown in FIG. 7.Again, blunt-ended sites (Z and W) are shown. In a first plasmid,containing the NER, a marker gene is inserted within the NER andoptionally a multiple cloning site is inserted within the marker gene.The foreign gene with its promoter, contained in a separate plasmid, isthen inserted into the marker gene by combining the two plasmids asshown. The foreign gene is inserted at any site within the marker genewhich will interrupt or change its marker function, whereby theinsertion can be recognised. For example, the E. coli. lacZ gene can beused as the marker gene. Only the alpha-fragment need be inserted sincethe remaining elements of the gene required for processing are providedby the bacterial host. It is provided with the E. coli. promoter forlacZ, the lacZ start codon and, preferably, a multiple cloning sitewithin a few codons of the start, followed by sequence encoding thealpha fragment. When the alpha-fragment is interrupted by a foreigngene, conveniently insertable in the said multiple cloning site, theblue colonies given on an X-gal substrate are not ordinarily obtained.White colonies indicate a likely recombination plasmid.

The recombination vector could, on occasion, be further provided with atermination signal site to terminate transcription of mRNA, which mightbe useful for stabilising the mRNA when an early-acting promoter isused. This would be inserted downstream of the foreign gene. However, inmany cases the ordinary termination signals of the infecting poxvirus orthe signals for starting transcription of another, downstream, gene ofthe FPV will be adequate.

References herein to vectors other than FPV (or VV) mean any convenientprokaryotic or eukaryotic cloning vector appropriate for bulk productionof the construct within a suitable host. Prokaryotic vectors willordinarily be plasmids or phages. Suitable prokaryotic hosts includebacteria. Eukaryotic vectors such as those of fungi, yeasts and animalcells, e.g. SV40, can be employed if thought more convenient.

Although the recombination vector used will ordinarily be ofdouble-stranded DNA, it is possible to use single-stranded DNA for thehomologous recombination.

The recombination vector of the invention containing the NER, promoterand foreign gene then has to be "swapped over" for the "parent" FPV DNA.For this purpose, appropriate animal, preferably but not necessarilypoultry, cells are infected with the parent FPV, and the recombinationvector is then introduced into the cells. This process is carried out invitro. It is best not to use wild type parent FPV for obvious reasons.FPV can readily be attenuated (mutated to make it less virulent), by anyconventional method of attenuation.

Many different methods are available for detecting or selecting therecombinant viruses, and have been described for VV in a review articleof M. Mackett and G. L. Smith, J. General Virology 67, 2067-2082 (1986).Such methods are applicable in the present invention. One method is toenlarge the recombination plasmid to include along with the desiredforeign gene an additional marker gene as described above (FIG. 6).Alternatively, the insertion of the foreign gene can be detected by ahybridisation assay in which a labelled nucleotide sequencecomplementary to the foreign gene sequence is employed. Such assays arewell known.

The FPV recombinants possessing the foreign gene are then grown inappropriate cells. The NER of the invention are non-essential in chickenembryo fibroblast cells. It would be necessary to carry out routineexperiments to determine whether they are also usable for growth of FPVin other kinds of cell, e.g. chicken embryo skin cells, chickenfibroblasts, chicken embryo epithelial cells derived by conventionaltissue culture methods, principally trypsinisation of tissues or thechorioallantoic membrane (CAM) of embryonated chicken or turkey eggs.

For administration to birds as a vaccine, the recombinant virus can begiven to birds by any suitable method such as by aerosol, drinkingwater, oral, intramuscular injection or inoculation into the wing web.Ingredients such as skimmed milk or glycerol can be used to stabilisethe virus. It is preferred to vaccinate chicks 1 day old by aerosoladministration. A dose of from 10² to 10⁸ pfu, preferably 10⁴ to 10⁶pfu, of the recombinant FPV per bird is recommended in general.

While the invention is intended primarily for the treatment of chickens,it is potentially of interest in relation to the vaccination of otheranimals which might safely be inoculated with FPV. It is even possiblethat it might be considered safe to infect humans with FPV afterappropriate trials have taken place.

The following Examples illustrate the invention.

EXAMPLE 1

This Example relates to terminal inverted repeat NERs.

Virus Strain

The HP438 strain of fowlpox virus was obtained from Professors A. Mayrand H. Mahnel , Ludwig-Maximillians University, Munich. The HP438 strainhas been obtained from the pathogenic HP1 strain by 438 passages inchick embryo fibroblasts (CEFs) in tissue culture, A. Mayr et al.,Zentralblatt fur Veterinarmedizin B13: 1-13 (1966). The HP 444 strainused to obtain DNA for cloning was derived by 6 further passages in CEFcells.

Tissue Culture Medium

CEF cells were grown in 199 (Wellcome) medium, supplemented withPenicillin (200 U/ml), Streptomycin (200 μg/ml), Fungizone (2 μg/ml) and10% newborn calf serum.

Cell Culture and Viral DNA Purification

FPV strain HP444 was grown in primary chick embryo fibroblast (CEF)cultures and FPV DNA prepared as described by M. M. Binns et al.,Nucleic Acids Research 15, 6563-6573 (1987).

Identification of the Terminal Fragment

The genome of poxviruses is double-stranded DNA. As usual the twostrands are held together by hydrogen-bonding. However, at the ends ofthe genome of poxviruses the two strands are covalently linked.Topologically this means that the DNA is a continuous strand. When thegenome is cut into fragments with a restriction enzyme, internalfragments are topologically different from terminal fragments and behavedifferently. On heating (to separate the two strands) the internalfragments completely separate and they reanneal slowly (hardly at all ifone chills quickly). The terminal fragments, being covalently bonded,cannot separate very far spatially. On cooling they reanneal quickly bystarting at the middle and "zipping up". This is called `snap-back`.After heating and rapid cooling therefore, only the terminal fragmentscan be seen by gel electrophoresis. This method of identification of theterminal fragment, called "the snap back method," see L. C. Archard & M.Mackett, J. General Virol, 45, 61-63 (1979), was used here.

A BamHI restriction digest (10 μg) DNA was extracted once with phenol,the aqueous phase was extracted with diethyl ether to remove phenol andthe DNA in the aqueous phase was precipitated with ethanol. The DNApellet was dried and resuspended in 20 μl of TE (10 mM Tris HCl pH 7.5,1 mM, EDTA). The DNA solution (10 μl) was then denatured by the additionof 40 μl of 95% v/v deionized formamide in 50 mM Tris HCl pH 7.5 andincubated at 60° C. for 10 minutes. The sample tube was then rapidlycooled on wet ice containing NaCl for 10 minutes before analysing theDNA by agarose gel electrophoresis. The presumed terminal fragmentrenatured rapidly only in the native FPV DNA and was detected as anapproximately 6.3 kb band. There is only one BamHI--derived snap backfragment, as the BamHI site falls well within the TIRs.

Cloning of the End Fragment of FPV DNA

The terminal crosslinks of the BamHI fragment were digested with S1nuclease; using 100 μg of FPV DNA and 20 units S1 nuclease (BoehringerMannheim) in 250 μl of 30 mM sodium acetate, pH 4.5; 0.3M NaCl; 1 mMZNSO₄ ; 5% (v/v) glycerol at 37° C. for 20 minutes. To end-repair theDNA, it was then ethanol-precipitated and re-suspended in 100 μl oflegation buffer (50 mM Tris. HCl pH 7.5, 10 mM magnesium chloride, 10 mMDTT) containing 5 units of E. coli polymerase I Klenow fragment and 1unit of T₄ DNA polymerase and 0.025 mM each of dATP, dCTP, dGTP, anddTTP. This reaction was left for 1 hour at room temperature. It wasstopped by heating for 10 minutes at 70° C. and 20 μl thereof wasdigested with 10 units of BamHI (Boehringer Mannheim) in accordance withthe manufacturer's recommendations. 5 μl of this DNA was ligated to 20ng of BamHI/Sma I--cut plasmid pBGS19, B. G. Spratt et al., Gene 41,337-342 (1986), to make a total volume of 20 μl ligation buffercontaining 2 units of T₄ DNA ligase and 1 mM ATP, and incubated at 4° C.overnight and transformed into E. coli. Screening for recombinants whichcontained the end fragment was carried out with a radiolabelled FPVBamHI terminal fragment isolated from a gel, using standard proceduresas described by T. Maniatis et al. "Molecular Cloning: A LaboratoryManual", New York, Cold Spring Harbor Laboratory (1982) and Holmes andQuigley, Analytical Biochemistry 114, 193-197. A recombinant plasmid pB3was isolated.

Cloning of NERs

The three genes under test for non-essentiality were retained togetherin their natural sequence in a shortened modification of plasmid pB3,see FIG. 8. pB3 was shortened by removing the short unique sequence andthe repeated sequences (see FIG. 8) from the BamHI fragment. This wasdone by a digestion with SstI There is a SstI site in pBGS19 just beforethe BamHI site into which the FPV BamHI fragment was inserted. The SstIdigestion was carried out at 37° C. in a total of 20 μl of therecommended buffer, using 10 units of SstI. There are many SstI sites inthe repeated sequence, the last of which causes cutting at nucleotide4005. Thus, after cutting with SstI, the plasmid pB3 (9.8 kb) wasre-ligated in a 100 μl volume, to favor intramolecular ligation usingthe previously described ligation buffer, at 15° C. overnight, toproduce plasmid pB3ME (5.8 kb) which lacks 4.0 kb of the BamHI fragmentDNA. To select the 5.8 kb clones, rather than those which have lostshorter lengths of SstI- cut DNA, selected colonies were grown up andthen subjected to gel electrophoresis. To check that all the repeatswere removed, the plasmid was digested with BglII and the fragmentsanalysed by gel electrophoresis.

Preparation of a Plasmid Containing the VV 7.5K Promoter and the lacZGene

The VV 7.5K promoter is available in a plasmid pGS20 from Dr. GeoffSmith, Division of Virology, Dept. of Pathology, University ofCambridge, Addenbrooke's Site, Hills Road, Cambridge, England. Cambridgeand is described in M. Mackett, G. L. Smith and B. Moss J. Virology 49,857-864 (1984) and can be combined with E. coli. lacZ sequences as shownin the DNA sequence below. The resultant plasmid is designated pPGl.Other methods which could be used to combine the VV 7.5K promoter andlacZ sequence have been described in papers relating to VV. ##STR2##

Insertion of the VV 7.5K Promoter/lacZ Gene Construct into FPV NERs toMake Recombination Plasmids

The P7.5+lacZ fragment (about 3.6 kb) in 2 μl total volume of buffer wascut out of the ampicillin--resistant plasmid pPGl with 10 units ofBamHI. The ends of the DNA were then repaired with the Klenow fragmentof DNA polymerase to produce blunt-ended DNA molecules. Thus, thefragment was phenol-extracted with an equal volume of TE- saturatedphenol and ethanol- precipitated. It was resuspended in 20 μl ofligation buffer containing 5 units of Klenow fragment in 1 unit of T₄polymerase, containing 0.025 mM of each of dATP, dCTP, dTTP and dGTP andstood for 1 hour at room temperature. Similarly, the pB3ME (SpeI, NcoIand BglII) were also digested with BamII and repaired to produceblunt-ended sites. The P7.5+lacZ fragment was then blunt-end ligatedinto pB3ME as follows. Thus, the plasmid and fragment (20 ng each) wereligated in a total volume of 10 μl above described ligation buffercontaining 1 unit of T₄ DNA ligase and 1 mM ATP and incubated at 4° C.overnight and were transformed into E. coli. Blue colonies of X-galKanamycin plates were selected (PBGS 19 is a Kanamycin-resistant vector)and the DNA of several colonies was examined by the method of Holmes andQuigley, loc. cit. Each ligation produced two different results, i.e.the P7.5+lacZ could be ligated into pB3ME in either of two differentorientations. The pairs of constructs of each site were called pEFL2/4,pEFL5/6 and pEFL7/8. In pEFL, 2, 6 and 8, the P7.5+lacZ insert was inthe same orientation as the gene into which it was inserted i.e. left toright in pEFL2 (BglII, 26.0) and pEFL8 (SpeI, 11.5) and right to left inpEFL6 (NcoI, 13.9).

Recombination

The plasmids PEFL 2, 6 and 8 which contained the P7.5K promoter/laczconstruct in the orientations described were used in recombinationexperiments. The recombination has the following general stages:

1. Infect CEF cells with FPV, and add the recombination plasmid DNA inthe form of a pre-prepared calcium phosphate precipitate. Allow to growfor several days. (Recombination takes place)

2. Harvest cells and freeze-thaw three times (Virus is released fromcells).

3. Add this preparation to CEFs at various dilutions to produce plateswith about 500-1000 plaques per 10 cm dish (about 0.1% of these arerecombinant plaques).

4. Add Xgal overlay and visualise blue plaques.

5. Pick isolated plaques. Freeze-thaw three times to release virus fromcells of plaque.

6. Titrate again on CEFs and get more plaques.

7. Loop back to 4, repeating stages 4-6, until a pure plaque of therecombinant is obtained.

In more detail, chick embryo fibroblast cells, when about 80% confluent,were infected with about 3 plaque forming units (infectious virusparticles) per cell of plaque-purified HP 440. fowlpoxvirus. The viruswas contained in 1 ml of commercially available serum free medium("199"), and was left to adsorb to the cells for 2 hours at 37° C.,after which the virus inoculum was removed and 10 ml of fresh mediumcontaining 5% newborn calf serum (CS) was added. 1.5 hours later aprecipitate of the plasmid DNA construct to be recombined into the viruswas prepared: to 1 ml of Hepes-buffered saline (pH7.12) was added 10-20μg of the pEFL 2, 6 or 8 plasmid DNA in solution in TE at about 1 μg/μl.50 μl of 2.5M calcium chloride was added slowly, and a fine DNA/calciumphosphate precipitate was allowed to form for 30 minutes at roomtemperature (25° C.). The medium from the virus-infected cells was thenremoved, the 1 ml of precipitate was added to the cells and left for 30minutes at room temperature. Then 10 ml of fresh medium containing 5% CSwas added to the cells, and they were incubated at 37° C. for a further4 hours. The condition of the cells was then examined and if the DNAcalcium phosphate precipitate was considered too thick, the medium wasreplaced. Otherwise it was then replaced the next day. The cells wereincubated at 37° C. for a further 5 days and then the virus washarvested by scraping the cells into the medium. Intracellular virus wasreleased by three cycles of freeze-thawing and the virus titrated by aconventional plaque titration.

The liquid medium was removed from the cells and an overlay of cellculture medium in 1% low gelling temperature agarose (LGT) containing300 μg/ml (5-bromo-4-chloro-3-indolylbeta D-galactoside) was added.After overnight incubation at 37° C., plaques expressing the lacZ gene,blue in colour, were observed.

Blue plaques were picked into 500 μl of 10 mM Tris pH9, intracellularvirus was released by three cycles of freeze-thawing and the virustitrated by a conventional plaque titration. The process of pickingplaques and re-titrating was repeated until a plaque purifiedrecombinant was obtained.

The plaque-purified recombinants were then passaged in tissue culturefor the following numbers of passages after the first picking:

    ______________________________________                                        recombinants from plasmid pEFL 8                                                                 (SpeI insertion): stable for at                                               least 3 passages                                           6                  (NcoI insertion): stable for                                                  only 2 passages                                            2                  (BglII insertion): stable for                                                 at least 6 passages.                                       ______________________________________                                    

It is possible that the NcoI-based recombinants might be stable for alonger period under different conditions from those tried above.

DNA Analysis of FPV Recombinants

When the DNA of the FPV parent is digested with BamHI a number ofrestriction fragments are generated, which can be separated on agarosegels. The terminal fragment is of size 6.3 kb and is of double intensitycompared with the other bands since there are two copies of it (one fromeach end of the genome). When the DNA of the recombinant made with pEFL2was digested the pattern of bands was the same except that the double6.3 kb fragment had been replaced by a double 9.9 kb fragment. This isconsistent with the increase in size from 6.3 kb to 9.9 kb which wouldbe produced if the 3.6 kb P7.5+lacZ fragment had been inserted into theterminal fragment. Probing of the restriction digest after transfer tonitrocellulose showed that the 9.9 kb band did in fact contain lacZsequences.

EXAMPLE 2

This Example relates to D8-D9 NERs.

In order to test whether D9, D10 and the fowlpox gene present in the D8position were non-essential for growth further manipulations werecarried out.

1. For D8 the plasmid pMB389 was grown in a dam⁻ E. coli host WK262 andreisolated as pMB434. The reason for this is that in the usual strainsof E. coli, the enzyme BclI to be used here cannot restrict because itscontrol adenine base in its TGATCA recognition sequence is methylated.The dam⁻ strain resists methylation and in this host the enzyme BclIwould be able to cleave its two recognition sites which occurred in theD8 region. This plasmid was then cleaved with BclI and religated and aclone from which the 583 bp BclI fragment was missing was isolated byscreening a number of colonies with small plasmid preps. Such a plasmidpMB441 containing a unique BclI site and having much of its D8 regionremoved was isolated. A BamHI fragment from pBPGl, containing theβ-galactosidase gene, under the control of the vaccinia P7.5K promoter,was then inserted into this unique BclI site of pMB441 (BamHI and BclIgenerate compatible sticky ends such that no end-repairing wasnecessary). A plasmid clone pMB447 containing the correct structure wasisolated and used in recombination assays (see later).

2. For D9 pMB389 was cleaved with AsuII (a unique site for which existsin the D9 gene) and end-repaired. pBPGl was cleaved with BamHI andend-repaired and then ligated with AsuII cleaved pMB389 which had alsobeen end-repaired. One colony selected as blue on Xgal Amp plates wasfound to contain the vaccinia P7.5K promoter and β-galactosidase geneinserted in the D9 gene.

3. For D10 an EcoRI fragment was subcloned from pMB389 (one EcoRI sitelies within the D9 gene whilst the other is in the polylinker of thepUC13 vector of pMB389) into pUC13 cut with EcoRI. The resulting plasmidpMB439 contains a unique BUIII site within the DIO gene. The BamHIfragment containing the vaccinia P7.5K promoter and the β-galactosidasegene were cloned into the BglII site of pMB439 to generate pMB443.(Again no end-repair was necessary as BamHI and BglII also havecompatible sticky ends). Plasmed pMB443 was then tested in recombinationassays.

The plasmid constructions described above are illustrated schematicallyin FIG. 10. The plasmid pBGI is derived by cutting the BamHI fragmentfrom pPGI, this fragment containing the P7.5-β-galactosidase geneconstruct, and inserting it in the plasmid pBGS19 (refer to Example 1).

Recombination

Viruses from initial blue plaques from a recombination experiment wereplaque-purified three times and appeared stable (i.e. no white plaqueswere observed on the third passage). Viral DNA was made from theplaque-purified viruses, and digested with EcoRI. DNA from the original(non-recombinant) ("wild type") FPV was also digested with EcoRI toserve as a control. The DNAs were run on duplicate agarose gels,Southern blotted, and probed with either the putatively recombinant FPVEcoRI fragment, within which the insertions should have taken place, orwith pPGl containing the β-galactosidase gene. Comparing the putativelyrecombinant FPV with the wild type when the EcoRI digests were run ongels, the normal EcoRI fragment present in wild-type DNA had increasedin size in both the D8 and D9 putative recombinants. The new D8 fragmentwas smaller than the new D9 one, as expected, due to the removal of thesmall BclI fragment (377-960) in this case. When probed withβ-galactosidase gene-containing pBGl plasmid DNA, wild-type FPV DNA didnot light up but the D8 and D9 putative recombinants both lit up in thesame positions as when the probe from the putatively recombinant EcoRIfragment was used. This established that the structures of the D8 and D9recombinants were as expected.

Similar experiments for the D10 gene failed to give the desiredconfirmations.

EXAMPLE 3 Virus Strain

This Example describes the construction of vectors containing NewcastleDisease Virus (NDV) F and HN genes, successful protection of birds usingthe F. gene. TIR NERs were used.

A. Subcloning of NDV Fusion (F) and Haemagglutinin-Neuraminidase (HN)Genes

The F and HN genes of NDV were provided as a gift from Professor P. T.Emmerson of the University of Newcastle-upon-Tyne, each cloned into theplasmid pUC19, P. Chambers et al., Journal of General Virology, 67,2685-2694 (1986); N. S. Millar et al . , Journal of General Virology,67, 1917-1927 (1986). See also European Patent Application 227414(National Research Development Corporation) which refers to BudapestTreaty patent deposits of NDV cDNA in plasmids pUC18, and pUC19 at theNational Collection of Industrial Bacteria, Aberdeen, Scotland on 1stJuly 1986 as NCIB 12277 (F gene) and 12278 (HN gene). Referring to thepUC19 clones actually used herein, the F gene clone also contained partof the HN gene downstream thereof. The F gene was subcloned by cuttingwith the restriction enzyme BamHI (which conveniently cuts 29 basesupstream of the initiating ATG codon at the 5' end of the F gene) andHindIII (which cuts approximately 820 bases downstream from the 3' endof the F gene, this extra sequence including some of the HN gene) intothe kanamycin-resistant plasmid pBGS18 cut with BamHI and HindIII. Theresulting plasmid was called pBGFl (see FIG. 11).

The pUC19 μlasmid containing the HN gene also has F gene sequenceupstream of the HN gene. In order to facilitate positioning of apoxvirus promoter adjacent to the HN gene, the F gene sequences wereremoved by the use of the exonuclease Bal31. The plasmid was cleavedwith the restriction enzyme SphI (which cuts in the multiple cloningsite of pUC19 on the 5' side of the HN gene) and digested for a range ofappropriate times as follows. The method used was as described in"Molecular Cloning (A Laboratory Manual)" by T. Maniatis et al., ColdSpring Harbor USA (1982). 20 μl of DNA (at approximately 500 μg/ml) weremixed with 20 μl BSA (1 mg/ml) and 40 μl of 2×"Bal31 buffer" (24 mMCaCl₂, 24 mM MgCl₂, 0.4M NaCl, 40 mM Tris/HCl pH 8.0, 2 mM EDTA) andwarmed to 30° C. 0.5 unit of Bal31 was added and samples were incubatedfor 0, 1, 2, 3, 4 and 5 minutes. Then 20 μl of water and 3 μl of 0.5MTris/HCl pH 7.5, 100 mM MgCl₂ were added, and the samples were digestedwith 10 units of SstI (which cuts downstream of the HN gene) for 60minutes. Samples were run on an agarose gel to estimate the amount ofDNA digested, and the 0, 1 and 2 minute samples were chosen for furtheruse. The molecules were then repaired (as described above) with T4 DNApolymerase and Klenow fragment and cloned into SmaI-cut pBGS18. Severaldifferent isolates were sequenced at the 5' end of the HN gene and onewas found in which only 49 of the bases upstream of the gene remained(starting at the sequence GGCTTCACAA . . . ). This plasmid was calledpBHNl (see FIG. 13).

B. Cloning of the F Gene into a Recombination Vector

In order to place the F gene under the control of the P7.5K VV promoter,the F gene was excised from pBGFl with BamHI and HindIII, end-repairedas described, and cloned into the Smai site of pGS20 (Mackett et al.,Journal of Virology 49, 857-864). This plasmid was called pGSFl (seeFIG. 11). A plasmid pEFL16 (FIG. 12) was constructed by inserting thePvuII fragment from pUC19 into the SpeI site of pB3ME (described above).This fragment contains the coding sequences of the alpha peptide ofbeta-galactosidase with a multiple cloning site arranged such that whenfragments are cloned into this site the translation of thebeta-galactosidase is disrupted and thus recombinants can be detectedbecause they are white colonies on plates containing Xgal. (Thisconstruct pEFL16 is now equivalent to the `recombination plasmid`described in Example 1 and shown in FIG. 7). The F gene plus P7.5promoter was excised from pGSF1 by using XbaI and NheI, end-repaired asdescribed, and cloned into the unique SmaI site of pEF16. The resultingplasmid (which now has the F gene, with the P7.5 promoter upstream,placed within the SpeI non-essential region of the terminal fragment offowlpox) is called pEFF2 (FIG. 12).

C. Cloning of the HN Gene into a Recombination Vector

In order to place the HN gene under the control of the P7.5 promoter itwas excised from pBHNl with SstI and BamHI, repaired as described, andcloned into the SmaI site of pGS20. This plasmid was called pGSHNl (FIG.13). The HN gene plus P7.5 promoter was excised from pGSHNl by usingSalI and Asp718, repaired as described, and cloned into pB3ME which hadbeen cut with SpeI and repaired. This plasmid was called pEFH3 (see FIG.14).

D. Production of FPV Recombinant Containing the F Gene

The Poxine strain of fowlpox virus used was kindly donated by Duphar. Itwas passaged three times in chicken embryo fibroblast cells CEFs intissue culture. A single plaque was picked at this stage and wasplaque-purified three times. The virus thus obtained was called Px4.1.and was grown in tissue culture as described for the HP444 strain inExample 1.

The plasmid pEFF2 was allowed to recombine into the Px4.1 strain of FPV,as described in Example 1. The virus from the recombinations wastitrated on CEFs and then plaqued out on 10 cm petri dishes at aconcentration of virus producing about 1000 plaques per dish. The viruswas overlaid with cell culture medium in 1% low gelling temperatureagarose. After 4-5 days the agarose overlay was removed and stored at 4°C. in a sterile petri dish. A dry nitrocellulose filter was laid ontothe cell sheet and pressed down with a circle of Whatman 3 MMchromatography paper soaked in 20 X SSC. The filter was then lifted offthe dish, with the cell sheet and plaques adhered to it, the 3MMs paperremoved, and the filter was baked at 80° C. in a vacuum for 2 hours. Toidentify recombinant viruses, the filters were then probed withradiolabelled probes specific for either the F gene or the HN gene. Thefilters were exposed to X-ray film and the position of recombinantplaques identified by aligning the autoradiograms with the storedagarose overlays. Recombinant virus was then isolated from the agaroseoverlay by removing a plug of agarose using a pasteur pipette. This plugwas resuspended in 10 mM Tris pH 9.0, freeze-thawed three times andreplaqued. Recombinant virus was identified again by probing, and thisprocess was repeated three times in all until a plaque-purified viruswas achieved.

E. Production of FPV Recombinant Containing the HN Gene

The plasmid pEFH3 was allowed to recombine, as described above, into theplaque-purified HP440 virus (HP 438 as described in Example 1, plus twofurther passages). This virus is called FP9. Recombinant viruscontaining the HN gene was isolated as described for the F gene.

F. Expression of the F Gene in Tissue Culture

The recombinant viruses, and the equivalent parent strain as controls,were used to infect 5 cm petri dishes of CEFs at approximately 5pfu/cell. The virus was allowed to grow for various times and then thecells lysed by addition of 500μl of boiling sample buffer (containing 24mM Tris/HCl PH 6.8, 1% SDS, 20% glycerol and 0.02% bromophenol blue).For the HN recombinant this buffer also included 0.1M dithiothreitol.The infected cells were scraped into the sample buffer, and the samplesboiled for 2 minutes, cooled on ice, and frozen at -20° C. Samples werethen loaded onto standard polyacrylamide protein gels, electrophoresedto separate the proteins, and transferred to nitrocellulose filters byWestern blotting. The Western blots were probed (as described in A. C.R. Samson et al., Journal of General Virology 67, 1199-1203) with amonoclonal antibody specific to the F protein or the HN protein did notreveal any F protein produced by the recombinant. The F protein producedby infection of CEFs with NDV was detected by the monoclonal antibody,but at a lower level than the HN, and so if the same ratio of native torecombinant protein held for the F as for the HN, the recombinant Fmight indeed not be seen.

G. Protection of Birds Against NDV Challenge

14 day old Rhode Island Red chickens were inoculated intravenously with10⁶ pfu/bird of the F gene recombinant (4 birds) or of a fowlpox strainFP9 having no inserted genes (6 birds). 6 birds were left uninoculated.The 10 birds not inoculated with the recombinant are hereinafterreferred to as `the control birds`. At 28 days the inoculation wasrepeated. At 64 days the birds were challenged intramuscularly with 10⁵×ELD₅₀ (ELD=embryo lethal dose) of the virulent Herts 33 strain of NDV.At 3 days after challenge, 4 of the control birds were dead and 8 werevery sick. At 4 days after challenge, 10 out of 12 control birds weredead, and by 5 days after challenge all the control birds were dead. All4 birds inoculated with the F recombinant were normal.

Serum was taken from birds inoculated with the F recombinant and controlbirds before challenge with NDV. Examination of this serum by probingWestern blots of purified NDV virions, showed that only the serum fromthe birds inoculated with the F recombinant had antibodies to the Fprotein. Thus although it was not possible to detect F protein producedby the recombinant in vitro, the in vivo experiment showed veryconvincingly that protection could be achieved, and that F protein insome form has been produced by the FPV recombinant containing the NDV Fgene.

Example 6 describes a protection experiment using the NDV HN geneconstructs of this Example.

EXAMPLE 4

This Example demonstrates vaccination of birds at one day of age with anNDV F gene/ FPV recombinant.

Groups of six or seven Rhode Island Red chicks were inoculated, at oneday of age, by the wing-web method with either the NDV F gene/poxine FPVrecombinant described in Example 3 or the Px4.1 parent poxine FPVstrain. Each virus strain was inoculated into four groups of birds, atdoses of 5×10⁴, 5×10³, 5×10², and 5×10¹ pfu/bird. Thus there were eightgroups in all, with an additional group of uninoculated birds. Birdswere inoculated at one day of age and challenged intramuscularly 26 dayslater, as in Application 8907374. Table 1 shows the results of thischallenge. Uninoculated and control birds were not protected againstchallenge, all 38 birds being dead by 4 days. The birds inoculated withthe fowlpox/NDV F recombinant were protected to a significant degree, 22birds out of 28 surviving the challenge (79%). The dose of vaccine usedappeared to have little effect on the levels of protection, although thelowest dose gave the best protection.

                  TABLE 1                                                         ______________________________________                                                         Total number of birds dead at                                                 days 3-6 after challenge with                                          Viral  NDV Herts 33 strain                                          Inoculum    dose-    days 3  day 4 day 5 day 6                                ______________________________________                                        NDV F gene/ 5 × 10.sup.4                                                                     0/7     0/7   2/7   2/7                                  P × 4.1 FPV                                                                         5 × 10.sup.3                                                                     0/7     0/7   2/7   2/7                                  recombinant 5 × 10.sup.2                                                                     1/7     1/7   2/7   2/7                                              5 × 10                                                                           0/7     0/7   0/7   0/7                                  P × 4.1 FPV alone                                                                   5 × 10.sup.4                                                                     6/7     7/7                                                          5 × 10.sup.3                                                                     3/6     6/6                                                          5 × 10.sup.2                                                                     5/6     6/6                                                          5 × 10                                                                           1/5     5/5                                              Uninoculated                                                                               --      4/7     7/7                                              ______________________________________                                    

EXAMPLE 5

This Example confirms the results given in Example 3 (Section G) forvaccination of birds with an NDV F gene recombinant and shows that itcan be done by using the wing web as well as the intravenous route.

The procedure of Example 3 was followed, except that the boosterinoculation was at 27 days from birth (rather than 28) and the challengeat 42 days from birth (rather than 64), the parent poxine strain wasused as control, and there were five groups of birds (two inoculated iv,two wing web and one uninoculated).

The results in Table 2 show that all the birds inoculated with the NDV Fgene/poxine FPV recombinant survived, whereas all the others died.

                  TABLE 2                                                         ______________________________________                                                        Total number of birds dead at                                                 days 3, 4 and 14 after challenge                                              with NDV Herts 33 strain                                      Inoculum    Method    day 3    day 4  day 14                                  ______________________________________                                        NDV F gene/                                                                   P × 4.1 FPV                                                             recombinant iv        0/6      0/6    0/6                                     P × 4.1 FPV alone                                                                   iv        6/6                                                     NDV F gene/                                                                   P × 4.1 FPV                                                             recombinant wing-web  0/6      0/6    0/6                                     P × 4.1 FPV alone                                                                   wing-web  4/6      6/6                                            Uninoculated                                                                               --        9/13    13/13                                          ______________________________________                                    

EXAMPLE 6

This Example shows that a NDV HN gene/FPV recombinant also givesprotection. Thus, protection can be obtained from either the F or the HNgene alone.

Example 5 was repeated except that the NDV HN/HP 440 FPV recombinant asdescribed in Example 3 (Section E) was used with the HP 440 strain ascontrol.

Table 3 shows the results. Again, the NDV NH gene-vaccinated birds wereprotected while the others all died.

                  TABLE 3                                                         ______________________________________                                                       Total number of birds dead at                                                 days 3, 4 and 14 after challenge                                              with NDV Herts 33 strain                                       Inoculum    Method   day 3     day 4 day 14                                   ______________________________________                                        NDV HN gene/                                                                  HP 440 FPV                                                                    recombinant iv       1/6       1/6   1/6                                      HP 440 alone                                                                              iv       6/6                                                      Uninoculated                                                                              --        9/13     13/13                                          ______________________________________                                    

EXAMPLE 7

In this Example recombination vectors were made using the Egogpt gene,the constructs being of a generally similar kind to those described byCSIRO in the already cited PCT Application WO 88/02022, but flanked bynon-essential region sequences of the present invention rather than TKgene sequences. The recombination vectors comprise in order:

(1) Non-essential region (around the Bgl III site)

(2) Ecogpt gene

(3) VV P.5 promoter

(4) VV P11 promoter

(5) IBV spike protein gene

(6) Non-essential region.

Note that the two promoters are "back-to-back", as shown in WO 88/02022,transcribing in opposite direction.

The clone pEFL2 (containing the vaccinia P7.5 promoter andbeta-galactosidase gene in the FPV terminal fragment, see above) was cutwith the restriction enzymes SphI and Ncol, repaired as described, andreligated. The resulting construct, designated pEFL18, had lostapproximately 850 base pairs of FPV sequence, as expected, but had alsolost a HindIII site immediately adjacent to the SphI site. Thus, thisplasmid contains only two HindIII sites, very close together, betweenthe P7.5 promoter and the beta-galactosidase gene. (Digestion withHindIII thus only removes a very small fragment and can be used for theinsertion of gene fragments containing their own ATG codon to makebeta-galactosidase fusion constructs). This plasmid was cut with HindIIIand Espl (Espl cuts near to the 3' end of the beta-galactosidase gene)and repaired. Into this repaired site was inserted a BglII/ApaI fragment(repaired) from the plasmid PSV2gpt (Mulligan & Berg, Science 209,422-1427, 1980) which contained the E.coli ecogpt gene. Thus, thebeta-galactosidase gene was removed and the Ecogpt gene was placed underthe control of the P7.5 promoter. A unique SalI site upstream of thepromoter allows insertion of other promoters or promoter/geneconstructs. This plasmid was called pEFGP13.

The P11 late vaccinia promoter was excised from pMM6 (a gift fromMichael Mackett) provided with multiple cloning sites (BamHI, XhoI, XbaIand SalI) by transfer to pUC19 and the resultant P1-cloning siteconstruct inserted into the SalI site of pEFGPT3. The resultant plasmidwas designated pEFGPT6 and allows insertion of genes at various sitesunder the control of the P11 promoter. An Infectious Bronchitis Virusspike protein gene (see NRDC's PCT Application publication No.WO86/05806) was inserted downstream of the promoter.

The plasmid pEFGPT3 was allowed to recombine into FPV strain FP9, asdescribed above. Recombinant viruses were selected by the use of MXHATmedium containing mycophenolic acid (Boyle & Coupar, Gene 65, 123-129,1988). After the initial recombination in the presence of MXHAT medium,the virus was released by freeze-thawing and passaged twice underselective conditions. A cytopathic effect was seen, indicating thatvirus was growing under the selective conditions. Virus was then plaquedout and probed with an Ecogpt-specific probe. A high proportion ofplaques (approximately 75%) were positive, indicating that the selectionhad been successful (normally only 0.1% of the virus would berecombinants) and hence that recombinants expressing the Ecogpt gene hadbeen isolated.

Similar recombination experiments were carried out to incorporate theEcogpt gene into the Poxine strain of FPV. After the recombination andupon passage in selective conditions, a cytopathic effect of the viruscould be seen, indicating that recombinant virus expressing the Ecogptgene had been produced. This confirms that the BglII non-essentialregion (as well as the SpaI region) is present in Poxine.

We claim:
 1. A recombinant fowlpox virus (FPV) which contains within thelong unique sequence of the terminal inverted repeat of the fowlpoxvirus genome, at least one foreign gene and at least one sequence forregulating the expression of the or each foreign gene.
 2. A cloningvector containing a fowlpox non-essential region (NER) sequence beinginterrupted by at least one foreign gene and at least one sequence forregulating the expression of the foreign gene, said NER sequence beinghomologously recombinable with sequence within the long unique sequence(LUS) of the terminal inverted repeat (TIR) of the fowlpox virus genome.3. A DNA molecule which consists essentially of a fowlpox non-essentialregion (NER) sequence being interrupted by at least one foreign gene andat least one sequence for regulating the expression of the foreign gene,said NER sequence being homologously recombinable with sequence withinthe long unique sequence (LUS) of the terminal inverted repeat (TIR) ofthe fowlpox virus genome.
 4. A recombination vector comprising a cloningvector, said cloning vector containing a fowlpox non-essential region(NER) sequence being interrupted by at least one foreign gene and atleast one sequence for regulating the expression of a said foreign gene,said NER sequence being homologously recombinable with sequence withinthe long unique sequence (LUS) of the terminal inverted repeat (TIR) ofthe fowlpox virus genome.
 5. A recombination vector according to claim 4wherein the NER sequence is homologously recombinable with sequencewithin 2.2 Kilobase pairs from the external end of the LUS.
 6. Arecombination vector according to claim 5 wherein the NER sequence ishomologously recombinable with sequence within an open-reading frame(ORF) which is substantially one of those at 4125-4790, 5261-5629 or5883-6185 of the nucleic acid sequence as shown in FIG. 15 or a variantthereof occurring in another strain of FPV.
 7. A recombination vectoraccording to claim 4, 5 or 6, which comprises in order:(1) a firsthomologously recombinable flanking sequence, (2) a first portion of saidNER sequence, (3) promoter DNA, (4) a foreign gene transcribablydownstream of the promoter whereby when the fowlpox virus RNA polymerasebinds to the promoter it will transcribe the foreign gene into mRNA, (5)a second portion of said NER sequence, the first and second portionsbeing in the same relative orientation as are the first and secondportions of the non-essential region within the viral genome and (6) asecond homologously recombinable flanking sequence.
 8. A recombinantfowlpox virus (FPV) which is the product of homologous recombination ofa parent FPV with a recombination vector claimed in claim 4, 5, or
 6. 9.An in vitro culture of animal cells infected with a recombinant FPVaccording to claim 8 or
 1. 10. A culture according to claim 9, whereinthe animal is a chicken.